Mechanism and therapy in DNM1 epileptic encephalopathy

脑细胞（神经元）通过释放化学神经递质进行交流。神经递质储存在称为突触囊泡（SVs）的神经元内的小球形隔室中。当神经元交流时，SVs与神经元外表面融合，导致神经递质释放。在神经递质释放后，这些sv通过一个称为内吞作用的过程进行改造。内吞过程中SVs的正确形成对于维持神经递质释放至关重要，因为SVs在神经元中高度受限。人们最初认为，这种重要过程的功能障碍会导致受影响个体的死亡，然而，在过去的10年里，现在越来越明显的是，内吞作用缺陷是一系列严重形式的癫痫的基础，称为癫痫性脑病（EEs）。就目前的情况来看，许多情感表达患者没有可用的治疗方法，这使得了解疾病机制成为开发新药必不可少的第一步。基因DNM1的突变是导致EE的一种形式。DNM1基因产生一种叫做动力蛋白-1的蛋白质，这种蛋白质对SV内吞作用至关重要。作为揭开内吞作用所必需的基因突变如何导致EE的第一步，我们制造了一只携带最常见的人类DNM1突变的小鼠。这只小鼠的神经元表现出功能失调的SV内吞作用，此外还有过度兴奋的大脑活动和癫痫样行为。因此，我们现在有了一个实验工具，通过它来确定癫痫是如何发展的，并在其中测试新药。在之前的工作中，我们发现了一种被批准用于人类的药物，可以加速SV内吞作用。我们发现这种药物也纠正了我们的小鼠DNM1 EE模型的所有缺陷（SV内吞，脑兴奋性和癫痫样事件）。由于该药很可能影响SV内吞作用而不是动力蛋白-1本身的功能，因此它有可能广泛应用于SV内吞作用的其他疾病。在这个工作计划中，我们将确定这种药物如何起作用，并在不同功能失调的SV内吞作用模型中测试其更广泛的治疗潜力。每个基因都有两个拷贝，称为等位基因。患有DNM1 EE的个体只有1个突变等位基因，另一个不受影响。有趣的是，人们可以忍受一个DNM1等位基因的完全丢失，只要另一个是功能性的，这表明1)突变形式通常会覆盖未受影响的版本的功能，2)去除突变等位基因可能是治疗这种疾病的一种有希望的方法。我们将通过使用基因疗法从小鼠模型中去除突变的DNM1等位基因来验证这一假设，以确定这是否纠正了所见的缺陷。确定突变的DNM1如何导致EE是至关重要的，因为它将为如何治疗这种疾病和其他相关疾病提供关键数据。上述详细介绍的基因治疗方法对于解决这个问题至关重要，因为我们可以从特定类型的神经元中去除突变的DNM1等位基因，以确定它是如何引起EE的。这对于确定癫痫机制至关重要。在DNM1基因中已经发现了许多不同的突变，因此确定它们在影响动力蛋白-1功能的方式上是否存在相似性以及它们如何改变神经元的功能是至关重要的。我们还将确定这些突变在其作用方面是否具有相似性，这将为未来的治疗提供大量信息。

Brain cells (neurons) communicate by releasing chemical neurotransmitters. Neurotransmitters are stored in small spherical compartments within neurones called synaptic vesicles (SVs). When neurones communicate, SVs fuse with the outer surface of the neuron causing neurotransmitter release. After neurotransmitter release, these SVs are reformed by a process called endocytosis. The correct formation of SVs during endocytosis is essential for the maintenance of neurotransmitter release, since SVs are highly limited in neurons. It was originally thought that dysfunction in such an essential process would result in death of the affected individual, however over the past 10 years it is now becoming apparent that defective endocytosis underpins a series of severe forms of epilepsy called epileptic encephalopathies (EEs). As things currently stand, there are no available therapies for many individuals with EE, making understanding disease mechanisms an essential first step in generating new drugs. Mutations in the gene DNM1, are causal in a form of EE. The DNM1 gene makes a protein called dynamin-1, which is essential for SV endocytosis. As a first step in unravelling how a mutation in a gene essential for endocytosis results in EE, we generated a mouse that carries the most common human DNM1 mutation. Neurons from this mouse displayed dysfunctional SV endocytosis, and in addition had over-excitable brain activity and seizure-like behaviour. Therefore we now have an experimental tool through which to determine how epilepsy develops and in which to test new drugs. In previous work we identified a drug approved for human use that accelerates SV endocytosis. We discovered that this drug also corrected all of the defects in our mouse model of DNM1 EE (SV endocytosis, brain excitability and seizure-like events). Because it is likely that this drug affects SV endocytosis rather than dynamin-1 function itself, it has potential to be widely used in other disorders of SV endocytosis. In this programme of work we will determine how this drug works, and test its wider therapeutic potential in different models of dysfunctional SV endocytosis. People have two copies of every gene, called alleles. Individuals that have DNM1 EE only have 1 mutant allele, the other is unaffected. Interestingly, people can tolerate the complete loss of one DNM1 allele providing the other one is functional, suggesting that 1) the mutant form usually overrides the function of the unaffected version and 2) removal of the mutant allele may be a promising approach to treat the disorder. We will test this hypothesis by using gene therapy to remove the mutant DNM1 allele from the mouse model to determine whether this corrects the defects seen. A determination of how mutant DNM1 causes EE is essential to obtain, since it will provide key data on how to treat this, and other related conditions. The gene therapy approach detailed above will be critical to address this question, since we can remove the mutant DNM1 allele from specific types of neurons to determine how it is causing EE. This will be critical in determining epilepsy mechanisms. Many different mutations in the DNM1 gene have been discovered, therefore it is essential to determine whether there are similarities in how they affect dynamin-1 function and importantly how they change the function of neurons. We will also determine whether these mutations have similarities in terms of their effect, which will be highly informative in terms of future therapies.